1. Amyotrophic Lateral Sclerosis (ALS; Lou Gehrig’s disease)

2. Lou Gehrig

3. ALS is a “motor-neuron” disease, a neurodegenerative disease characterized by the selective death/degeneration of upper motor neurons (in the brain) and lower motor neurons (in the spinal cord). The upper motor neurons normally send the signals to the lower motor neurons, which send signals to muscles.

6. A-myo-trophic Lateral Sclerosis
Amyotrophic: no muscle nourishment
Lateral: refers to the the areas in a person's spinal cord where portions of the nerve cells that signal and control the muscles are located
Sclerosis: scarring of the affected nerves

7. Severe atrophy of anterior spinal roots in motorneuron disease
Degeneration of motor neurons in the spinal cord and brainstem results in degeneration of pyramidal tracts and severe atrophy of anterior spinal roots which is demonstrated here.

8. Atrophic muscle fibers in ALS patients

9. Muscular atrophy in ALS

10. Symptoms Earliest signs:
1-twitching (fasciculation), stiffness, cramping
2-weakness of the arms and of the legs. This results in an increased frequency of stumbling on uneven pavement or difficulty in climbing stairs. Arm weakness may lead to difficulty in grasping or holding a cup, for instance, or loss of coordination in fingers
3-more rarely, weakness of the muscles of the mouth. This results in difficulties for the patient to chew, swallow and speak.
Later signs:
The progression of the disease is accompanied by weight loss, fatigue, exaggerated reflexes, and decreased coordination. Ultimately, patients cannot walk, stand, eat, or breathe without assistance. Increased susceptibility to pneumonia and respiratory failure causes half to die within three years.
However, muscles that controls eye movements and urinary sphincters are spared.

11. Diagnosis of ALS -No biological marker has been identified yet.
-Series of clinical and neurological exams
-MRI
-myelogram of cervical spine (an x-ray analysis that allows the detection of lesions in selected area of the spinal cord)
muscle and/or nerve biopsy
electromyography (EMG) and nerve conduction velocity (NCV), to measure muscle response to nervous stimulation.

12. MRI in Amytrophic Lateral Sclerosis (ALS)

13. Electromyography (EMG)
A needle electrode is inserted through the skin into the muscle. Each muscle fiber that contracts will produce an action potential. The presence, size, and shape of the wave form of the action potential produced on the oscilloscope, provides information about the ability of the muscle to respond to nervous stimulation.

14. Epidemiology of ALS
Incidence: 1-2 in 100,000 each year. At the moment in the U.S. there are 25,000 people affected by the disease. Median age of onset is 55 years old. Gender-related incidence: female:male ratio is 4:5.
10% of the cases are inherited, familial cases (FALS), whereas the 90% of the cases are sporadic (SALS)
The life-span of a patient affected by ALS is 3 to 5 years, after the diagnosis.

15. Genetic of ALS

16. Toxic mechanisms in ALS

17. Is ALS a mitochondrial disease?

18. Evidences of mitochondrial dysfunction in SALS:
-mitochondria aggregate in skeletal muscle and intramuscular nerve
-mitochondria show abnormalities in proximal axons and nerves of the anterior horn of spinal cord
-increased mitochondrial volume and calcium level
-dysfunction of mitochondrial complex I and IV
Decreased ATP production

19. Calcium deposits in mitochondria of ALS patients
CONTROL
ALS
Calcium deposits in mitochondria of ALS patients
Syklos et al.,

20. Evidences that SOD1 could be involved in SALS
-Symptoms and pathology of SALS patients are the same as in SOD1 related FALS patients
-Pathologic alterations of SOD1 mutant mice are similar to those observed in SALS patients
Formation of mitochondrial vacuoles
Expansion and rupture of the mitochondrial outer membrane
Alterations of calcium homeostasis in the mitochondria
Alterations of mitochondrial membrane potential
Formation of calcium deposits within the mitochondria

21. Superoxide Dismutase 1 SOD1
SOD1 is a ubiquitous mostly cytosolic protein
SOD1 is comprised of 153 aa with an approximate molecular weight of 16kDa and is an active homodimer
Each of the two dimers of SOD1 binds a Cu++ and a Zn++ ion. The reduction of Cu++ to Cu+ is behind the mechanism of SOD1 in regulating the dismutation of superoxide ion O2-. into hydrogen peroxide H2O2
Cu2+ + O2-· Cu+ + O2
Cu+ + O2-· + 2H+ Cu2+ + H2O2
2 O2-· + 2H+ H2O2 + O2
A catalase will subsequently reduce hydrogen peroxide to water.

22. SOD1 structure: the functional homodimer

23. Most common mutations in SOD1 related FALS

24. SOD1 misfolds and aggregates in FALS SOD1A4V motorneurons
SEDI Ab
SEDI-reactive SOD1
SOD1

25. Possible roles of mutated SOD1 in FALS
Loss of physiological function: impaired dismutase activity
Gain of toxic function:
1) Aberrant redox chemistry, probably due to changes in the conformation of SOD1, that leave the channel (the portion of the molecule accepting superoxide ion, i.e.) able to accept larger molecules. This can lead to peroxidation, tyrosine nitrosylation and reverse catalysis (due to improper binding of Zn++ to the molecule that leads to formation of superoxide ion rather than dismutase activity). These activities are not a characteristic of ALL SOD1 mutations, thus remain partially controversial.
2) Protein instability and SOD1 aggregation. These activities are characteristic of all SOD1 mutants.

26. Models of mutant SOD1-mediated toxicity

27. Mutant SOD1 may mediate cytotoxic reactions involving: 1) Copper catalysis/Zn-mediated toxicity 2) Protein aggregation
Mutant SOD1 may mediate cytotoxic reactions involving: 1) Copper catalysis/Zn-mediated toxicity 2) Protein aggregation 4 4 1 1
O-O…Cu Zn WT
O-O…Cu Zn WT H H O O Cu Cu Zn Zn H H O O 2 2 3 3
Aggregation
Aggregation
.OH
.OH
Peroxidation
Peroxidation Cu Cu
ONOO
ONOO Zn Zn
toxic
toxic Cu Cu Zn Zn
toxic
toxic
NO-Tyr
NO-Tyr
Cu, Zn Toxicity
Cu, Zn Toxicity
Tyrosine Nitration
Tyrosine Nitration
P.Pasinelli

28. SOD1 and FALS
-More than 125 mutations have been found on the SOD1 gene, 114 are related to ALS, most of them are missense mutations, only 12 are nonsense mutations or deletion mutants.
-Most mutations reduce dismutation activity, however others retain full dismutase activity, still are related to the disease. Moreover, there is NO CLEAR CORRELATION between enzyme activity and progression of the disease.
-In addition, in animal models, gene KO for SOD1 does not cause motorneuron disease, whereas overexpression of SOD1 does.
In this respect, the simple manipulation of SOD1 dismutase activity IS NOT necessarily behind ALS/motorneuron disease.

29. 1-SOD1 could be modified in SALS
2-SALS and FALS may share the same toxic mechanism of toxicity of SOD1

30. Can SOD1 form aggregates also in SALS?

32. wtSOD1 can be oxidized

33. SOD1 conformation-specific antibody

34. Conformation-specific SOD1 antibody detects
oxSOD1 in non-denaturing conditions…

35. …but not in denaturing conditions:
SOD1 oxidation as a mechanism to form SOD1 aggregates

36. SOD1 oxidation inhibits anterograde Fast Axonal Transport FAT

37. oxSOD1 affects the activity of p38MAPK, a protein that regulates FAT

38. Inhibition of p38MAPK reverts the effects of oxSOD1 on FAT

39. Sequestration of conformation-specific oxSOD1 reverts the effects
of wtSOD1 on FAT in SALS

40. Conformation-specific SOD1 antibody reacts with wtSOD1
only in SALS

41. How SOD1 aggregates could be toxic?
1- Formation of small and large aggregates that may impairs proteasomal activity. This would result in lack of proper degradation of different proteins including toxic mutant SOD1.
2- Sequestration within the aggregate of proteins that are important for the cell, like heat shock proteins (HSP70), thus impairing the physiological “protective” activity of these proteins.
3-SOD1 can sequester into aggregates the anti-apoptotic Bcl2.
4- Formation of SOD1 aggregates can be related to mitochondrial dysfunction and apoptosis.

42. SOD1 aggregates: mitochondrial dysfunction and apoptosis

43. 1- Cluster of abnormal mitochondria and morphological defect (in muscles cells and in motorneuron) Complex I and IV are defective, paralleled by increased mitochondrial Ca++ levels.
2-in vitro and in vivo, SOD1 mutants
*cause depolarization of mitochondria
*decrease levels of ATP
3- Vacuolated mitochondria: detachment between the outer and the inner membrane, crystae disruption.
Is mitochondrial dysfunction a cause or a consequence?

44. How could mutant SOD1 be related to mitochondrial defects?
Altering mitochondrial structure, causing formation of vacuoles and ultimately rupture of the mitochondrial outer membrane leading to apoptosis

45. SOD1 associates to Bcl2, but not to Bax, in vitro…
Exogenous SOD1
Endogenous SOD1

46. …and in vivo
mice
Human spinal cord

47. Bcl2 binds aggregated SOD1 in animal models of ALS…

48. …and in spinal cord of ALS patients FALS SOD1 A4V

49. Mutant SOD1 binds Bcl2 specifically in the mitochondria

50. SOD1 aggregates recruit Bcl2 and start apoptosis in FALS

51. More importantly:
Mutant SOD1 is very toxic when in the mitochondria
INSIDE THE MITOCHONDRIA
*forming aggregates
*interfering with cytochrome c in the peroxisomes (fusion of the peroxisomes membranes, formation of pores and release of cytochrome c, initiation of apoptosis)
OUTSIDE THE MITOCHONDRIA
*forming aggregates in the outer membrane, this leads to disruption of TOM (Translocator Outer Membrane complex) disruption of protein transport in the mitochondrion.
*forming aggregates with anti-apoptotic Bcl2 at the surface of the mitochondria, thus leading to apoptosis

52. The mitochondrion as a target of mutant SOD1

53. Defective axonal transport in ALS

54. Characteristic of ALS is axonal swelling

55. Axonal transport: Intracellular transport in neurons
-Neurons are polarized cells. The presence of a positive or negative pole provides driving force to regulate anterograde or retrograde transport.
-Anterograde: from cell body to neuronal end. Positive pole.
-Retrograde: from neuronal end back to cell body. Negative pole.
What is transported?
Proteins (synthesized in the cell body)
Organelles-Mitochondria
Vesicles

56. Axonal Transport

57. Proteins regulating axonal tranport
Kinesin 1 Anterograde transport
Dynein/dynactin Retrograde transport

58. Point mutations in the gene of dynactin are associated to FALS

59. ALS and axonal transport
In motorneurons, axons are very long thus both anterograde and retrograde transport are complex events
Neuronal transport is reduced in ALS
Deposition of neurofilament
Defects and mutations in dynein

60. Deposition of fibrillar proteinacious material
in Amyotrophic Lateral Sclerosis (ALS)
Ross and Poirier, 2004

61. Axonal transport in ALS
-Increased anterograde and decreased retrograde axonal transport in ALS patients.
-Dynactin mutations associated to impaired retrograde transport
-Decreased transport of mitochondria also in certain SOD1 mutants

62. Mechanisms of axonal transport defects: damage to mitochondria

63. Axonal vacuolation is an early event that precedes neurodegeneration in a model of ALS

64. Aggregates of G93ASOD1 are found in the axons, co-localizing
with dynein: possible role of mutant SOD1
in damaging axonal transport
Neuroreport

65. Only G93ASOD1, not WT SOD1, forms aggregates in the axon
that co-localize with dynein

66. Model of aberrant interaction between
SOD1 aggregates and dynein on FALS

67. What about autophagy in ALS?

68. Autophagy increases in SOD1G93A mutant mice
Activation of the autophagy marker LC3 follows the disease progression

69. Mitochondrial dysfunction and number of autophagic vacuoles follows the progression of the disease in SOD1G93A mutant mice

70. The expression of the autophagy marker LC3 does not change in ALS animal model

71. What about HSP that can regulate autophagy in ALS?

72. HspB8 is more abundant in the spinal cord of ALS mice

73. Mutant SOD1 co-localizes with HspB8 in ALS mice

74. HspB8 increases autophagic markers in ALS mice

76. SOD1 mutation in ALS: gain of toxic function rather than
loss of physiologic function

77. SOD1siRNA improves ALS symptoms and viability in a SOD1G93A mutant mouse.